AU2020387891B2

AU2020387891B2 - Humanized 4-1BB monoclonal antibody and pharmaceutical composition thereof

Info

Publication number: AU2020387891B2
Application number: AU2020387891A
Authority: AU
Inventors: Qing Li; Fanxin Ma; Zhong Wang
Original assignee: Sound Biopharmaceuticals Co Ltd
Current assignee: Sound Biopharmaceuticals Co Ltd
Priority date: 2019-11-19
Filing date: 2020-11-13
Publication date: 2024-11-07
Anticipated expiration: 2040-11-13
Also published as: JP2023501744A; EP4063388A4; US20220403039A1; EP4063388A1; AU2020387891A1; CN117843786A; JP7522480B2; CN112898426A; WO2021098597A1

Abstract

The present invention discloses a humanized 4-1BB monoclonal antibody, antigen binding fragment, pharmaceutical composition and medical use thereof. The monoclonal antibody comprises: a heavy chain CDR1 selected from the amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:4, a heavy chain CDR2 selected from the amino acid sequence shown in SEQ ID NO: 2 and SEQ ID NO: 5, a heavy chain CDR3 of the amino acid sequence shown in SEQ ID NO: 3, a light chain CDR1' of the amino acid sequence shown in SEQ ID NO: 6, a light chain CDR2' of the amino acid sequence shown in SEQ ID NO: 7, and a light chain CDR3' of the amino acid sequence shown in SEQ ID NO: 8. The monoclonal antibody can specifically bind to human 4-1BB, leading to increased T cell proliferation and TNF-γ production, which enhances and stimulates the immune response mediated by human 4-1BB.

Description

Humanized 4-1BB monoclonal antibody and pharmaceutical

composition thereof Technical Field

[0001] The present disclosure relates to a humanized 4-1BB monoclonal antibody, an antigen-binding fragment thereof, a pharmaceutical composition and a medical use

thereof.

Background

[0002] 4-1BB, also known as CD137 or TNFRSF9, belongs to the TNFR superfamily with a molecular weight of 50-55 kDa and is expressed on T cells, NK cells, NK T cells,

dendritic cells (DCs), Tregs, and PDCA+ B cells. 4-1BB binds to the ligand 4-1BBL

expressed on APC cells with high affinity, thereby promoting T cell proliferation,

enhancing cytokine production, and preventing activation-induced cell death (AICD).

[0003] Agonistic anti-4-1BB monoclonal antibodies (mAbs) have significant

inhibitory effects on various autoimmune diseases and viral infections. A number of

studies have shown that 4-1BB plays an important role in the pathogenesis of

rheumatoid arthritis (RA). Soluble forms of 4-1BB and 4-1BB ligand (4-1BBL) are

higher in RA patients than in healthy individuals, and their levels correlated with disease

severity. In 4-1BB antibody treated arthritic mice, there was robust expansion of a novel

CD8 T cells subset co-expressing the CDli marker. These newly developed

CDllc+CD8+ T cells are demonstrated to be responsible for the reduced arthritis

symptoms. Further analysis revealed that the anti-4-1BB-induced CDllc+CD8+ T cells

expressed high levels of IFN-y. Experimental autoimmune uveoretinitis (EAU) is an

inflammatory disease of the vascular layer of the eye that leads to visual impairment and

can result in total blindness. Studies have shown that co-administration of 4-1BB

antibody with an EAU-inducing agent (inter-photo receptor retinoid-binding protein,

IRBP) results in significant expansion of CDllc+CD8+IFN-y+ T cells and IDO* DCs,

and these cells in combination destroyed the pathogenic CD4+ T cells. In addition,

4-1BB antibodies were also used to treat other autoimmune diseases, including multiple

sclerosis (MS), type 1 diabetes (TID), and lupus. Studies have also found that 4-1BB antibodies can interfere with certain viral infections, such as HSV-1, Japanese encephalitis virus (JEV), vaccinia virus, and lymphocytic choriomeningitis virus

(LCMV).

[0004] The in vivo anti-tumor effect of 4-1BB antibodies were first discovered by Melero et al. in 1997. These authors observed that 4-BB antibodies inhibited the

growth of the poorly immunogenic sarcoma and highly immunogenic in mice.

Subsequently, several investigators established that 4-1BB antibodies, either on their

own or in combination with other anti-tumor agents, have powerful anti-cancer

properties. When 4-1BB antibodies were injected into tumor-bearing SCID mice, tumor

growth was significantly inhibited. Treatment of mice harboring sarcoma or glioma cells

with 4-1BB antibodies prolonged the survival of the mice and led to tumor regression in

a T cell-dependent manner. Furthermore, the 4-1BB antibodies have also shown

therapeutic effects in endothelial tumors, renal cell tumors, and lung cancers. In addition,

numerous studies have shown that 4-1BB antibodies are more effective when they are

used in combination with other anti-cancer agents, for example, a combination of 4-1BB

antibody and PD-i antibody, a combination of 4-1BB antibody and CTLA-4 antibody, a

combination of 4-1BB antibody and GM-CSF, a combination of 4-1BB antibody and

CD40 antibody, a combination of cisplatin and 4-1BB antibody, a combination of

cyclophosphamide and 4-1BB antibody, a combination of 4-BB antibody and

cytokine-induced killer cell, a combination of 4-1BB antibody and CD4+ T cell

depletion, a combination of IL-2 and 4-1BB antibody, and etc. When used in

combination with other anti-cancer agents, the anti-tumor effect mediated by 4-1BB

antibody is amplified, which provides new ideas for the treatment of cancer. Variants of

the 4-1BB antibody, such as the ScFv of the 4-1BB antibody, also have significant

anti-tumor effects. Intra-tumoral injection of anti-4-1BB ScFv inhibited the growth of

Hepa 1-6 tumors, and this anti-tumor effect is dependent on increased IFN-y and

increased tumor infiltration by T cells.

[0005] However, no 4-1BB antibody has thus far been approved, and many clinical

studies failed, of which unsatisfactory effect in vivo and serious side effects including

hepatotoxicity, thrombocytopenia, leukopenia, etc., were the main reasons for their failure. Currently, only two 4-1BB mAbs are in clinical trials, fully humanized Urelumab from Bristol Myers Squibb and PF2566 from Pfizer. Therefore, there is a need to provide more 4-1BB antibodies to meet the market demand.

Summary

[0006] The present disclosure provides a humanized 4-1BB monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody comprises (1) heavy

chain complementarity determining regions CDR1, CDR2, CDR3, wherein the CDR

comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, the CDR2

comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5, and the

CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; and (2) light chain

complementarity determining regions CDR1', CDR2', CDR3', wherein the CDR1'

comprises the amino acid sequence shown in SEQ ID NO: 6, the CDR2' comprises the

amino acid sequence shown in SEQ ID NO: 7, and the CDR3' comprises the amino acid

sequence shown in SEQ ID NO: 8.

[0007] Particularly, the present disclosure provides a humanized 4-1BB monoclonal

antibody or antigen-binding fragment thereof, wherein a heavy chain variable region of the

humanized 4-1BB monoclonal antibody is the heavy chain variable region contained in the

amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12, and a

light chain variable region of the humanized 4-1BB monoclonal antibody is the light chain

variable region contained in the amino acid sequence shown in SEQ ID NO: 11.

[0008] In another aspect, the present disclosure provides a humanized 4-1BB monoclonal

antibody or an antigen-binding fragment thereof, wherein monoclonal antibody comprises

a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence

shown in SEQ ID NO: 12 or SEQ ID NO: 13, and the light chain comprises the amino acid

sequence shown in SEQ ID NO: 14.

[0009] In another aspect, the present disclosure provides a pharmaceutical composition

comprising the humanized monoclonal antibody or antigen-binding fragment thereof

disclosed herein, and a pharmaceutically acceptable carrier.

[0010] In another aspect, the present disclosure provides a kit comprising an independent

first formulation comprising the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein and an independent second formulation with anticancer activity. The second preparation is, for example, PD-i antibody, CTLA-4 antibody, GM-CSF, CD40 antibody, cisplatin, cyclophosphamide, cytokine-induced killer cells, IL-2, etc.

[0011] In another aspect, the present disclosure provides uses of the humanized monoclonal antibody or antigen-binding fragment thereof that binds to human 4-1BB disclosed herein in the manufacture of a medicament for treating cancer, autoimmune diseases, inflammatory diseases or viral infections.

[0012] The humanized monoclonal antibodies or antigen-binding fragments thereof disclosed herein have broad medicinal uses, such as for the treatment of cancer, autoimmune diseases, inflammatory diseases or viral infections. In one aspect, the present disclosure provides a method of treating cancer in a subject, comprising administering a therapeutically effective amount of the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein that binds to human 4-1BB to the subject. In another aspect, the present disclosure provides a method of treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein that binds to human 4-1BB to the subject. In another aspect, the present disclosure provides a method of treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein that binds to human 4-1BB to the subject. In another aspect, the present disclosure provides a method of treating a viral infection in a subject, comprising administering a therapeutically effective amount of the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein that binds to human 4-1BB to the subject.

[0013] In addition, the present disclosure also provides cell lines producing the antibodies of or antigen-binding fragments thereof disclosed herein, recombinant expression vectors comprising the nucleotides disclosed herein, and methods producing antibodies by culturing the antibody-producing cell lines.

[0014] The humanized monoclonal antibody disclosed herein binds to human 4-1BB with high affinity and specificity, leads to a significant increase in T cell proliferation and TNF-y production, and enhances and stimulates human 4-1BB-mediated immune response. The antibodies can be used as immunopotentiators for antitumor or antiviral immune responses, or as immunomodulators for T cell-mediated autoimmune diseases.

The antibodies can also be used as diagnostic reagents to detect human 4-1BB in the

blood or tissues of patients with cancer, autoimmune diseases or other diseases.

Brief Description of the Drawings

[0015] Figure 1. Generation of mAbs against rh4-1BB. A. schematic illustration of hybridoma generation and screening. B. ELISA data of serum titration assay. The

binding intensity (OD 4 50 ) was shown as the mean of triplicate wells. C. Binding affinity

measurement results of mAbs to 4-1BB antigen.

[0016] Figure 2. 4-1BB antibodies can bind cell membrane bound 4-1BB. Flow

cytometry analysis of 4-1BB mAbs on activated T cells.

[0017] Figure 3. ELISA to evaluate antibody 4-1BB ligand competition activity. A. Binding activity of mAbs to 4-1BB in the presence of certain amount of 4-1BB Ligand

previously immobilized on ELISA plates. B. Schematical representation of 4-BB

recognized by two different kinds of antibody.

[0018] Figure 4. MP4-1and 2D10 inhibited tumor growth in vivo. NOD/SCID mice (n

= 5 per group) were engrafted subcutaneously with LS174T cells (1x106 per mouse) and

freshly isolated human PBMCs (5x106 per mouse), then treated intraperitoneally with

vehicle (PBS, Black), MP4-1 (5 mg/kg, Red) or 2D10 (5 mg/kg, Blue) as described

above. The tumor volume was then measured. The data represent the average tumor

volume of five mice. The error bars represent the standard error (*** P<0.001, Dunnett's

multiple comparisons test, vehicle vs MP4-1 and vehicle vs 2D10).

[0019] Figure 5. Characterization of humanized MP4-1 candidates. A. Kinetic

interactions of mAbs with 4-1BB determined by BLI analyses. B. Binding activity of

mAbs to 4-1BB in the presence of 4-BB Ligand previously immobilized on ELISA

plates.

[0020] Figure 6. PP9150 can specifically bind activated T cells. Flow cytometry

analysis of 4-1BB mAbs on Jurkat cells (A) and activated T cells (B).

Detailed Description of the Embodiments

Antibodies

[0021] As used herein, the term "antibody" as used herein refers to any form of antibody that exhibits the desired biological activity, for example, inhibiting the binding

of a ligand to its receptor or inhibiting receptor signal transduction induced by the

ligand. "Antibody fragment" and "antigen-binding fragment" refer to antigen-binding

fragments of antibodies and antibody analogs, typically including at least a portion of

the antigen-binding or variable regions (e.g., one or more CDRs) of the parent antibody.

Antibody fragments retain at least some of the binding specificity of the parent antibody.

Typically, antibody fragments retain at least 10% of the binding activity of the parent

antibody when the activity is expressed on a molar basis. Preferably, antibody fragments

retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity

of the parent antibody for the target. Examples of antibody fragments include, but are

not limited to: Fab, Fab', F(ab') 2, and Fv fragments; diabodies; linear antibodies;

single-chain antibody molecules, such as sc-Fv; nanobodies; domain antibodies; and

multi-specific antibodies formed from antibody fragments.

[0022] As used herein, the term "Fab fragment" consists of one light chain and the

CHI and variable regions of one heavy chain. The heavy chain of a Fab molecule

cannot form a disulfide bond with another heavy chain. The "Fc" region contains two

heavy chain fragments comprising the CHI and CH2 domains of the antibody. The two

heavy chain fragments are held together by two or more disulfide bonds and by

hydrophobic interactions of the CH3 domains. A "Fab' fragment" contains a light chain

and a portion of a heavy chain comprising the VH and CHI domains and the region

between the CHI and CH2 domains, whereby an interchain disulfide bond can be

formed between the two heavy chains of two Fab' fragments to form a F(ab') 2 molecule.

A "F(ab') 2 fragment" contains two light chains and two heavy chains comprising

portions of the constant region between the CHI and CH2 domains, wherein an

interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab') 2

fragment consists of two Fab' fragments held together by disulfide bonds between the

two heavy chains. A "Fv region" comprises variable regions from both heavy and light chains, but lacks constant regions.

[0023] As used herein, the term "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In general, Fv polypeptides contain an additional polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0024] As used herein, the term "diabody" refers to small antibody fragment with two antigen-binding sites. The fragment comprises a heavy chain variable region (VH) linked to a light chain variable region (VL) in the same polypeptide chain, e.g., VH-VL or VL-VH. By using a linker that is short enough to pair two domains that are not on the same chain, the domains are forced to pair with the complementary domains of the other chain and form two antigen binding sites.

[0025] As used herein, the term "human antibody" refers to an antibody whose amino acid sequence corresponds to that of an antibody produced by a human, and/or an antibody that has been prepared using any of the techniques for preparing human antibodies shown herein. Humanized antibodies that contain non-human antigen-binding residues are specifically excluded from this definition. Monoclonal antibodies

[0026] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible natural mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and can be directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on an antigen. The modifier "monoclonal" refers to the properties of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method to prepare said antibody. For example, the monoclonal antibodies disclosed herein may be prepared by hybridoma or recombinant DNA.

Monoclonal antibodies may include "chimeric" antibodies.

[0027] In one aspect, the 4-BB monoclonal antibody disclosed herein comprises (1) heavy chain complementarity determining regions CDR1, CDR2, CDR3, wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; and (2) light chain complementarity determining regions CDR1', CDR2', CDR3', wherein the CDR1' comprises the amino acid sequence shown in SEQ ID NO: 6, the CDR2' comprises the amino acid sequence shown in SEQ ID NO: 7, and the CDR3' comprises the amino acid sequence shown in SEQ ID NO: 8.

[0028] In some embodiments, the complementarity determining regions of the 4-1BB monoclonal antibody comprise (a) CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, CDR1' comprises the amino acid sequence shown in SEQ ID NO: 6, CDR2' comprises the amino acid sequence shown in SEQ ID NO: 7, and CDR3' comprises the amino acid sequence shown in SEQ ID NO: 8; or (b) CDR comprises the amino acid sequence shown in SEQ ID NO: 4, CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5, CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3, CDR1' comprises the amino acid sequence shown in SEQ ID NO: 6, CDR2' comprises the amino acid sequence shown in SEQ ID NO: 7, and CDR3' comprises the amino acid sequence shown in SEQ ID NO: 8.

[0029] In another aspect, the 4-1BB monoclonal antibody disclosed herein comprises (1) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10; and (2) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 11.

[0030] In another aspect, the 4-1BB monoclonal antibody disclosed herein comprises (1) a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13; and (2) a light chain comprising the amino acid sequence shown in SEQ ID NO: 14.

[0031] It is expected that the binding domain of the monoclonal antibody disclosed herein may carry a signal peptide, which is usually located at the N-terminus of the secreted protein and generally consists of 15-30 amino acids. When the signal peptide sequence is synthesized, it is recognized by the signal recognition granule (SRP), protein synthesis is suspended or slowed down, the signal recognition granule carries the ribosome to the endoplasmic reticulum, and the protein synthesis restarts. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity, and the signal peptide sequence is cleaved under the action of the signal peptidase. If the termination transit sequence exists at the C-terminus of the nascent peptide chain, it may not be cleaved by the signal peptidase, for example, ovalbumin contains an internal signal peptide, and neither its precursor nor the mature form is cleaved by signal peptidase. An exemplary signal peptide sequence is MDPKGSLSWRILLFLSLAFELSYG. Another exemplary signal peptide sequence is METDTLLLWVLLLWVPGSTG.

[0032] As used herein, the term "specifically binds" means that the monoclonal antibodies disclosed herein are capable of specifically interacting with at least two, three, four, five, six, seven, eight or more amino acids of each human target molecule. The "specific binding" of an antibody is mainly characterized by two parameters: a

qualitative parameter (binding epitope or antibody binding site) and a quantitative parameter (binding affinity or binding strength). Antibody binding epitopes may be determined by FACS, peptide dot epitope mapping, mass spectrometry, or peptide ELISA. The Biacore and/or ELISA may measure the binding strength of an antibody to a specific epitope. Signal-to-noise ratios are often calculated as a representative measure of binding specificity. In such a signal-to-noise ratio, the signal represents the strength of antibody binding to the target epitope, and the noise represents the strength of antibody binding to other non-target epitopes. Preferably, when the signal-to-noise ratio for a target epitope is about 50, the evaluated antibody may be considered to bind to the target epitope in a specific manner, i.e., "specifically binds". Variants

[0033] As used herein, a "variant" sequence refers to a sequence that differs from the sequence shown at one or more amino acid residues but retains the biological activity of the resulting molecule.

[0034] As used herein, the terms "conservatively modified variants" or "conservative amino acid substitutions" refer to amino acid substitutions known to a skill person in the art that generally do not alter the biological activity of the resulting molecule. In general, it is recognized by a skilled person in the art that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity.

[0035] As used herein, "% identity" between two sequences refers to a function of the number of identical positions shared by the sequences, i.e., % identity = number of equivalent positions/total number of positions x 100, wherein the number of gaps and the length of each gap will be considered, and the gaps need to be introduced when performing an optimal alignment of the two sequences. Sequences alignment and determination of % identity between two sequences may be accomplished using mathematical algorithms. For example, the % identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller (Comput.Appl.Biosci., 4: 11-17(1988)). This algorithm has been introduced into the ALIGN program (version 2.0), which uses the PAM120 weight residue table with a gap length penalty of 12 and a gap penalty of 4. Additionally, the % identity between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)). This algorithm has been introduced into the GAP program of the GCG package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0036] When referring to a ligand/receptor, antibody/antigen, or other binding pair, "specific" binding refers to a binding reaction that determines the presence or absence of

a protein in a heterogeneous population of the protein and/or other biological agent. Thus, under the specified conditions, a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts. Humanized Antibodies

[0037] Humanized antibodies have one or more amino acid residues from a non-human source. Humanization is generally carried out by substituting rodent CDRs or CDR sequences for the corresponding sequences of the human antibody. Thus, the "humanized" antibodies are chimeric antibodies in which a very small portion of the fully human variable domains have been replaced with corresponding sequences from non-human species. In practice, humanized antibodies are generally human antibodies in which certain CDR residues and possibly certain FR residues are replaced by residues from analogous sites in non-human (e.g., rodent) antibodies.

[0038] The selection of human variable domains (both light and heavy chains) used to prepare humanized antibodies is extremely important to reduce antigenicity. According to the so-called "best fit" method, the variable domain sequences of rodent antibodies are screened against the complete library of known human variable domain sequences. The human sequence closest to the rodent sequence was then used as the human framework (FR) for the humanized antibody. Another approach uses a specific framework derived from sequences common to all human antibodies of a specific subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies.

[0039] More importantly, the antibody is humanized so that it retains high affinity for the antigen and other favorable biological properties. To this end, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptually humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to a skilled person in the art. Computer programs are available that elucidate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these revealed structures allows analysis of the possible role the residues play in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the acceptor and import sequences so as to obtain desired antibody properties, such as increased affinity for one or more target antigens. In general, CDR residues are directly and most fully involved in influencing antigen binding.

[0040] Humanization of antibodies is simple protein engineering. Almost all murine antibodies can be humanized by CDR grafting, thereby maintaining antigen binding.

Alternatively, it is now possible to generate transgenic animals (e.g., mice) capable of

producing a complete repertoire of human antibodies after immunization without the

production of endogenous immunoglobulins. For example, it has also been

demonstrated that homozygous deletion of joining region (JH) gene of the heavy chain

of the antibody in chimeric and germline mutant mice completely inhibits production of

endogenous antibody.

AntibodyPurification

[0041] When using recombinant techniques, antibodies may be produced

intracellularly, in the periplasmic space, or secreted directly into the medium. If the

antibody is produced intracellularly, particulate debris (host cells or lysed fragments) is

removed as a first step, e.g., by centrifugation or ultrafiltration. When the antibody is

secreted into the medium, the supernatant from the expression system is typically first

concentrated using a commercially available protein concentration filter, e.g., Amicon

or Millipore Pellicon ultrafiltration units. Protease inhibitors (e.g., PMSF) may be used

in any of the preceding steps to inhibit proteolysis and antibiotics may be used to

prevent the growth of foreign contaminants.

[0042] Depending on the antibody to be recovered, other protein purification

techniques may also be used, for example, fractionation on ion exchange column,

ethanol precipitation, reverse phase HPLC, silica gel chromatography, anion or cation

exchange resin (e.g., polyaspartic acid column) chromatography, chromatographic

focusing, SDS-PAGE and ammonium sulfate precipitation. In one embodiment, glycoproteins can be purified by a method comprising: adsorbing the glycoprotein to a

lectin substrate (e.g., lectin affinity column) to remove the fucose-containing

glycoprotein from the preparation and thereby enrich the fucose-free glycoprotein.

Pharmaceutical Compositions and Kits

[0043] "Pharmaceutical composition" refers to a pharmaceutical formulation for use in humans. The pharmaceutical composition comprises humanized monoclonal antibodies or antigen-binding fragments thereof disclosed herein and suitable formulations of carriers, stabilizers and/or excipients. The present disclosure provides pharmaceutical formulations comprising the monoclonal antibodies or antigen-binding fragments thereof disclosed herein. To prepare a pharmaceutical composition or a sterile composition, the antibody or antigen-binding fragment thereof is mixed with a pharmaceutically acceptable carrier or excipient. The therapeutic and diagnostic preparation of drugs can be prepared in the form of, for example,lyophilized powder, slurry, aqueous solution or suspension by mixing with physiologically acceptable carriers, excipients or stabilizers.

[0044] The toxicity and therapeutic efficacy of antibody compositions administered alone or in combination with immunosuppressive agents can be measured in cell

cultures or experimental animals by standard pharmaceutical methods, such as methods

for determining LD 5 0 (dose causing 50% of the population lethal) or ED5 0 (dose

effective to treat 50% of the population). The dose ratio between toxic and therapeutic

effects is the therapeutic index, which can be expressed as the ratio of LD5 0 to ED5 0

. The data obtained from these cell culture assays and animal studies can be used to

formulate a range of dosages for use in humans. The dosage of the antibody is

preferably within a range of circulating concentrations that include the ED5 0 with little

or no toxicity. The dosage can be varied within this range according to the dosage form

used and the route of administration used.

[0045] Suitable routes of administration include parenteral administration (for example, intramuscular, intravenous or subcutaneous administration) and oral administration. The

antibody used in the pharmaceutical composition or for practicing the method of the

present invention can be administered in a variety of conventional ways, such as oral

ingestion, inhalation, topical application or transdermal, subcutaneous, intraperitoneal,

parenteral, intraarterial or intravenous injection. In one embodiment, the antibody of the

invention is administered intravenously. In another embodiment, the antibody of the

invention is administered subcutaneously. Alternatively, one can administer the antibody

in a local rather than systemic manner (usually a long-acting or sustained-release formulation), for example via injection of the antibody directly to the site of action. In addition, one can administer the antibody in a targeted drug delivery system.

[0046] The appropriate dose is determined by the clinician, for example, using parameters or factors known or suspected to affect the treatment or expected to affect the treatment in the art. Generally, the starting dose is slightly lower than the optimal dose, and thereafter a small increase until the desired or optimal effect relative to any adverse side effects is achieved. Important diagnostic measures include measuring, for example, inflammatory symptoms or the level of inflammatory cytokines produced.

[0047] The antibodies, antibody fragments and cytokines can be administered by continuous infusion or by dosing at regular intervals, for example, one day, one week, or 1-7 times a week. The dose can be provided intravenously, subcutaneously, intraperitoneally, transdermally, topically, orally, nasally, transrectally, intramuscularly, intracerebrally, intraspinally, or by inhalation. A preferred dosage regimen is a regimen that includes the maximum dosage or dosing frequency that avoids significant undesirable side effects. The total weekly dose is usually at least 0.05 ptg/kg body weight, more usually at least 0.2 pg/kg, most usually at least 0.5 pg/kg, typically at least 1 g/kg, more typically at least 10 pg/kg, most typically at least 100 pg/kg, preferably at least 0.2 mg/kg, more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg, ideally at least 10 mg/kg, more ideally at least 25 mg/kg, and most ideally at least 50 mg/kg. Based on mol/kg calculation, the required dose of small molecule therapeutics such as peptide mimetics, natural products or organic chemical agents is approximately the same as the dose of antibodies or polypeptides.

[0048] The pharmaceutical composition disclosed herein may also contain other agents, including but not limited to cytotoxic agents, cell growth inhibitors, anti-angiogenic drugs or antimetabolites, targeted tumor drugs, immunostimulants or immunomodulators, or antibodies conjugated to cytotoxic agents, cell growth inhibitors or other toxic drugs. The pharmaceutical composition can also be administered with other treatment modalities such as surgery, chemotherapy, and radiation. Typical veterinarians, experiments or research subjects include monkeys, dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

[0049] The humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein can be used alone or in combination with the following substances:

anti-tumor drugs or immunogenic agents, such as attenuated cancer cells; tumor

antigens including recombinant proteins, peptides and carbohydrate molecules;

antigen-presenting cells, such as tumor-derived dendritic cells stimulated by the antigen

or nucleic acid, immunostimulatory cytokines (such as IL-2, IFN 2 , GM-CSF) and cells

transfected with genes encoding immunostimulatory cytokines (such as but not limited

to GM-CSF); standard cancer treatment (such as chemotherapy, radiotherapy or surgery);

or other antibodies, including but not limited to antibodies against the following

antigens: VEGF, EGFR, VEGF receptors, other growth factor receptors, CD20, CD40,

CTLA-4, OX-40, 4-IBB and ICOS.

[0050] Accordingly, the present disclosure provides a kit for carrying out the above-mentioned combination therapy, which comprises an independent first

formulation comprising the humanized monoclonal antibody or antigen-binding

fragment thereof disclosed herein and an independent second formulation with

anticancer activity. In some embodiments, the subject is sometimes administered the

second formulation concurrently with the humanized monoclonal antibody or

antigen-binding fragment thereof disclosed herein. In some embodiments, the second

formulation and the humanized monoclonal antibody or antigen-binding fragment

thereof disclosed herein are administered separately. In some embodiments, the second

formulation or other agent typically administered to cancer patients and the humanized

monoclonal antibody or antigen-binding fragment thereof disclosed herein may be

administered in combination as a pharmaceutical composition.

[0051] As used herein, the term "second formulation" or "second anti-cancer agent" refers to any anti-tumor drug, including but not limited to: PD- antibody, CTLA-4

antibody, GM-CSF, CD40 antibody, cisplatin, cyclophosphamide, cytokine-induced

killer cells, IL-2, etc.

[0052] In one aspect, the present disclosure provides a method of treating a tumor

comprising administering a therapeutically effective amount of a humanized

monoclonal antibody or antigen-binding fragment thereof disclosed herein to a subject suffering from a tumor. In some embodiments, the humanized monoclonal antibody comprises (1) heavy chain complementarity determining regions CDR1, CDR2, CDR3, wherein the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, the CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 5, and the CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; and (2) light chain complementarity determining regions CDR1', CDR2', CDR3', wherein the CDR1' comprises the amino acid sequence shown in SEQ ID NO: 6, the CDR2' comprises the amino acid sequence shown in SEQ ID NO: 7, and the CDR3' comprises the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the humanized monoclonal antibody comprises (1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 10; and (2) a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 11. In some embodiments, the humanized monoclonal antibody comprises (1) a heavy chain H comprising the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13; and (2) a light chain L comprising the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, the subject is a mammal, preferably a human.

[0053] In another aspect, the present disclosure provides a method of treating a tumor comprising administering a therapeutically effective amount of a pharmaceutical composition disclosed herein to a subject suffering from a tumor. In some embodiments, the pharmaceutical composition comprises a humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein and a second anti-cancer agent. In some embodiments, the subject is a mammal, preferably a human. Treatment

[0054] When "administering" and "treating" refer to an animal, human, subject, cell, tissue, organ or biological fluid, it refers to contacting the animal, human, subject, cell, tissue, organ or biological fluid with an exogenous drug, therapeutic agent, diagnostic agent or composition. "Administration" and "treatment" may refer to, for example, therapeutic methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treatment of the cells includes contacting the agent with the cells and contacting the agent with a fluid, wherein the fluid is in contact with the cells. "Administration" and "treatment" also mean in vitro and ex vivo treatment of cells, e.g., by agents, diagnostic agents, binding compositions, or by other cells.

[0055] As used herein, "inhibition" or "treatment" includes delaying the development of symptoms associated with a disease and/or reducing the severity of these symptoms that the disease will or expected to develop. The term also includes alleviating existing symptoms, preventing additional symptoms, and alleviating or preventing the underlying causes of these symptoms. Therefore, the term means that a beneficial result has been conferred on a vertebrate subject suffering from a disease. Therapeutically Effective Amount

[0056] As used herein, the term "therapeutically effective amount" or "effective amount" refers to when humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein is administered alone or in combination with another therapeutic agent to a cell, tissue or subject, it effectively prevents or slows the amount of the disease or condition to be treated. A therapeutically effective dose further refers to the amount of the antibody sufficient to cause alleviation of symptoms, such as treating, curing, preventing or alleviating related medical conditions, or improving the treatment rate, cure rate, prevention rate, or alleviation rate of the symptoms. When administered to an individual alone, the therapeutically effective amount refers to the mount of the alone ingredient. When a combination is administered, the therapeutically effective amount refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether it is administered in combination, sequentially or simultaneously. A therapeutically effective amount will reduce symptoms usually by at least 10%; usually at least 20%; preferably at least about 30%; more preferably at least 40% and most preferably at least 50%.

[0057] In the present disclosure, "about" means that the value is within an acceptable error range of the particular value determined by one of ordinary skill in the art, wherein the value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" can mean within 1 or more than 1 standard deviation in the art. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean at most one order of magnitude or at most five times the value. Unless otherwise indicated, when a specific value appears in the application and in the claims, the meaning of "about" or "substantially comprising" should be assumed to be within an acceptable error range for the specific value.

Cancer

[0058] The humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein can be used to treat cancers, i.e., inhibit the growth or survival of tumor

cells. Preferred cancers whose growth can be inhibited by the antibody disclosed herein

include cancers that generally respond to immunotherapy. Non-limiting examples of

preferred cancers for treatment include, but are not limited to, esophageal cancer,

stomach cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, breast

cancer, cervical cancer, corpus carcinoma, ovarian cancer, bladder cancer, head and

neck cancer, endometrial cancer, osteosarcoma, prostate cancer, and neuroblastoma.

Autoimmune Disease

[0059] As used herein, the term "autoimmune disease" refers to a class of diseases

induced by damage caused by attacking its own organs, tissues or cells due to the

breakdown of immune tolerance of the immune system of body to its own components.

This can be limited to certain organs or involve specific tissues in different locations.

Treatment of autoimmune diseases is usually with immunosuppression, such as drugs

that reduce the immune response.

[0060] In the present disclosure, the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein can be used to treat autoimmune

diseases. Autoimmune diseases that can be treated with the antibodies or

antigen-binding fragments thereof disclosed herein include, but are not limited to,

Graves' disease, multiple sclerosis, autoimmune liver disease, primary adrenal atrophy,

chronic thyroiditis, type 1 diabetes, systemic lupus erythematosus, psoriasis, Crohn's

disease, atopic dermatitis, autoimmune hemolytic anemia, myasthenia gravis, demyelinating disease, eczema, graft-versus-host disease, rheumatoid arthritis, scleroderma, sjogren syndrome, chronic nephritis, ankylosing spondylitis, chronic active hepatitis, atrophic gastritis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome, idiopathic thrombocytopenic purpura, idiopathic leukopenia, chronic thyroiditis, pernicious anemia, chronic ulcerative colitis. Inflammatory Disease

[0061] As used herein, the term "inflammatory disease" refers to a disease caused by, produced by, or resulting in inflammation. The term "inflammatory disease" can also refer to a dysregulated inflammatory response that results in an overreaction of macrophages, granulocytes, and/or T lymphocytes, resulting in abnormal tissue damage and/or cell death. Inflammatory diseases can be acute or chronic inflammatory conditions and can be caused by infectious or non-infectious causes.

[0062] In the present disclosure, the humanized monoclonal antibody or antigen-binding fragment thereof disclosed herein can be used to treat inflammatory diseases. Inflammatory diseases that can be treated with the antibodies or antigen-binding fragments thereof disclosed herein include, but are not limited to, arthritis, tendinitis, arteriosclerosis, polymyalgia rheumatica, bursitis, cystic fibrosis, arthrosteitis, giant cell arteritis, polymyositis, dermatomyositis, pemphigus, pemphigoid, mixed connective tissue disease, sclerosing cholangitis, inflammatory bowel disease, ulcerative colitis, inflammatory skin diseases, asbestosis, silicosis, pneumoconiosis, sarcoidosis, extrinsic allergic alveolitis, hepatitis, delayed type hypersensitivity, pneumonia, respiratory tract inflammation, adult respiratory distress syndrome (ARDS), encephalitis, immediate hypersensitivity, asthma, hay fever, allergies, acute anaphylaxis, rheumatic fever, cystitis, chronic cholecystitis, allograft rejection, host-versus-graft rejection, appendicitis, arteritis, bronchiolitis, bronchitis, cervicitis, cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis, endocarditis, endometritis, enteritis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastroenteritis, gingivitis, iritis, laryngitis, myelitis, myocarditis, nephritis, omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, mumps, pericarditis, pharyngitis, pleurisy, phlebitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, orchitis, tonsillitis, urethritis, uveitis, vaginitis, vulvitis, vasculitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and cardiovascular inflammation.

Viral Infection

[0063] As used herein, the term "infection" refers to the invasion of organisms by pathogens, their proliferation, and the response of host tissues to these organisms and

the toxins they produce. Infections can be caused by infectious agents such as viruses,

viroids, prions, bacteria, nematodes (such as parasitic roundworms and pinworms),

arthropods (such as ticks, mites, fleas and lice), fungi (such as ringworm), and other

giant parasites such as tapeworms and other worms. In the present disclosure, the

infectious agent is a virus.

[0064] "Viral infection" refers to the process by which a virus invades the body through various pathways and proliferates in susceptible host cells. In the present

disclosure, the humanized monoclonal antibody or antigen-binding fragment thereof

disclosed herein can be used to treat viral infections. Viruses causing the viral infection

include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV),

human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2

(HIV-2), metapneumovirus, parainfluenza virus, influenza virus, respiratory syncytial

virus (RSV), adenovirus, rhinovirus, coronavirus, enterovirus, coxsackievirus, dengue

virus, Japanese encephalitis virus (JEV), hepatitis A virus, hepatitis B virus, hepatitis C

virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpes virus type

8, measles virus, mumps virus, human papilloma virus, polio virus, rabies virus, rubella

virus and varicella-zoster virus.

[0065] Diseases or conditions associated with the viral infection include, but are not

limited to: retinitis, enteritis, infectious mononucleosis, Hodgkin's lymphoma, Burkitt's

lymphoma, nasopharyngeal carcinoma, Acquired Immune Deficiency Syndrome

(AIDS), Upper Respiratory Tract Infection (URI), Lower Respiratory Tract Infection

(LRI), myocarditis, encephalitis, Dengue Haemorrhagic Fever/Dengue Shock Syndrome

(DHF/DSS), hepatitis A, hepatitis B, hepatitis C, gingivostomatitis, keratoconjunctivitis,

skin papules, mumps, polio, rabies, rubella and chickenpox.

Immune Adjuvants

[0066] The humanized monoclonal antibodies or antigen-binding fragments thereof of the disclosure can be used in combination with other recombinant proteins and/or

peptides (e.g., tumor antigens or cancer cells) to increase the immune response to these

proteins (i.e., in a vaccination regimen). For example, a humanized monoclonal

antibody or antigen-binding fragment thereof can be used to stimulate an

antigen-specific immune response by co-administering the humanized monoclonal

antibody or antigen-binding fragment thereof and an antigen of interest, e.g., a vaccine.

Accordingly, in another aspect, the present disclosure provides a method of enhancing

the immune response of a subject to an antigen, comprising administering (i) the antigen;

and (ii) the humanized monoclonal antibody or antigen-binding fragment portion

thereof disclosed herein to the subject to increase the immune response to the antigen.

For example, the antigen can be a tumor antigen, a viral antigen, a bacterial antigen, or

an antigen from a pathogen. Non-limiting examples of such antigens include, but are not

limited to, tumor antigens or antigens from viruses, bacteria, or other pathogens.

Combinational Therapy

[0067] As described above, the monoclonal antibody or antigen-binding fragment

portion thereof disclosed herein can be co-administered with one or more other

therapeutic agents, such as cytotoxic agents, radiotoxic agents or immunosuppressive

agents. The antibody can be conjugated to the agent as an immune complex, or can be

administered separately from the therapeutic agent. In the latter case (separate

administration), the antibody can be administered before, after or concurrently with the

therapeutic agent, or it can be co-administered with other known therapies.

[0068] The antibodies can also be used in in vivo diagnostic assays. The antibody is usually labeled with a radionuclide, such as mIn, 99Tc, 4C , 1 I, 3H, 3P, 35S, or "F, so

that immunoimaging or positron imaging can be used to locate the antigen or

antigen-expressing cells.

[0069] The present disclosure will be more fully understood by referring to the

following examples. However, these examples should not be construed as limiting the

scope of the present disclosure. All documents and patent citations mentioned herein are expressly incorporated herein by reference.

Examples

[0070] Example 1. Generation of mAbs against rh4-1BB

[0071] Animals

[0072] All animal experiments were carried out under the authority of the Animal Experiment Facility of Sun Yat-sen University. Balb/c mice were purchased from the

Animal Experiment Facility of Sun Yat-sen University. Non-obese diabetic-severe

combined immunodeficiency (NOD/SCID) mice were purchased from Charles River

Laboratories. Animals were housed in barrier rooms with pathogen-free conditions for

those experiments involving immune-compromised mice.

[0073] Productionof monoclonal antibody

[0074] To generate mAbs against rh4-1BB, four weeks old female Balb/c mice

immunized subcutaneously (S.C) with human 4-1BB extracellular domain protein (Acro

Biosystems) at 3 times with two weeks' intervals. All mice were bled one week after the

third time's injection, sera were collected for screening via indirect ELISA to determine

immune response. The splenocytes from immunized mice were separated and fused

with SP2/0 mouse myeloma cells (purchased from the Shanghai Cell Bank) following

standard procedure. Cells were seeded into 96-well plates and maintained in culture in

hybridoma growth medium: RPMI-1640(Gibco by life technology) containing 20%

FBS, 1x HAT media supplement (Sigma). Hybridoma supernatants were screened by

indirect ELISA and positive wells were selected for cloning (by limiting dilution

process). Stable monoclones were selected for expansion and characterization of

antibodies.

[0075] Result: In our earlier attempt to produce anti-4-1BB antibody, we used a higher

dosage (50 ug per mouse) when do primary immunization, but all mice died within 10

days. As human 4-1BB showed 60% identity of amino acid sequence to mouse 4-1BB

[2], probably the rh4-1BB served as a stimulated molecule and induce strong immune

response that cause mice death. Then we decreased the dosage to 30 ug per mouse

followed with two time's boosting immunization. To test the immunization efficacy,

serum collected from mice before immunization and after the third injection were tested via indirect ELISA. The serum of the immune mice showed positive absorbance (OD >

0.2) at 1/8000 dilution (Figure IB). Hybridoma clones were then generated and

screened by ELISA using 4-1BB protein. 5 different hybridoma clones with strong

binding were obtained and chose for further analysis.

[0076] Example 2.4-1BB binding affinity of mAbs

[0077] Affinity determinationby Biolayer interferometry (BLI)

[0078] Antibody affinity for 4-1BB extracellular domain was measured using an Octet RED96 instrument (ForteBio, Pall Life Sciences). All assays were performed with

agitation set to 1000 rpm in assay buffer (PBS, PH7.4, 0.02%(v/v) Tween 20). Assays

were performed in solid black 96-well plates (Greiner Bio-one, 655209) at 30°C.

Purified 4-1BB antibodies in PBST were loaded onto the surface of anti-mouse IgG Fv

Capture Biosensors (AMQ). Then a biosensor baseline step was used before the analysis

of association of the antibody on the biosensor to the testing antigen for 300s. Testing

antigen was developed into a twofold concentration gradient in a titration series of

seven. Following is a dissociation interaction recorded in wells containing assay buffer.

Baseline drift was corrected by subtracting the shift recorded for a sensor loaded with

antibody by not incubated with antigen. Octet data were evaluated using data analysis

software version 9.0 (PALL/ForteBio) and a global fit 1:1 modal was used to determine

the KD value.

[0079] Flow cytometry analysis of antibodies binding to cell surface 4-BB

[0080] 0.5 ug/ml OKT3 pre-coated in flat bottom 6-well plates and 0.5 ug/ml soluble

PFC-1 (IL-15 fusion protein) were used to stimulate purified T cells at 37C in a 5%

CO 2 humidified incubator. After 7 days' stimulation, activated T cells were harvested.

4-1BB antibodies were incubated with activated T cells or Jurkat cells (purchased from

the Shanghai Cell Bank). Then cells were washed, and cell-bound antibody was

detected with Alexa Fluor 488-labeled goat anti-human or mouse IgG (H+L) specific

antibody and analyzed on a FC500 flow cytometer (BECKMAN COULTER).

[0081] Result: To evaluate the binding of mAbs to 4-1BB, the affinity of different

antibodies to antigen was measured. MP4-1 showed superior binding to recombinant

human 4-1BB by BLI analysis. The observed equilibrium dissociation constant (KD) for

4-1BB ECD was 0.0865 nM, which is more than 35-fold higher than the other

mAb-candidates (Figure IC). Binding of mAbs to cell membrane bound 4-1BB was

measured using flow cytometry (FACS) with stimulated primary human T cells

described above. Same as the positive control PF2566, each of our 4-BB antibodies

showed binding with activated T cells (Figure 2).

[0082] Example 3. Antibody 4-1BB ligand competition activity

[0083] Antibody 4-JBB ligand competition assay

[0084] To determine if 4-1BB antibodies can block the interaction of 4-1BB and its ligand, the 4-1BB ELISAs were carried out. Briefly, the extracellular domain of human

4-1BB Ligand (4-1BBL) protein with Fc tag (Acro Biosystems) was coated on ELISA

plates, followed incubation with human 4-1BB protein (His tag) in the presence of

4-1BB antibodies. HRP-linked anti-6X His tag antibody (Abcam) was used to detect

4-1BB protein bound on its ligand. Then TMB substrate solution (TIANGEN) was used

for detection. Samples were analyzed at OD 4 5 0 /6 2 0 using a microtiter plate reader

(infinite F50, TECAN).

[0085] Statisticalanalysis

[0086] Statistical significance was done by GraphPad Prism 7.0 software (GraphPad

Software, La Jolla California USA). Statistical analysis was performed by one-way

ANOVA followed by Dunnett's multiple comparisons test was employed. *P<0.05,

**P<0.01, ***P<0.001 and data represent mean SEM unless otherwise noted.

[0087] Result: Antibodies were tested for their ability to block the binding of the

human 4-1BB protein to plate bound recombinant 4-1BB ligand using ELISA.

Consistent with previous report, PF2566 bound competitively with recombinant human

4-1BB ligand (4-1BBL) to recombinant human 4-1BB (Figure 3A). For our 4-BB

antibodies, MP4-1 and 10A3 can block the interaction of 4-1BB and its ligand with

different ability, while 2D10 and 9B4 can't affect the interaction (Figure 3A). Obviously,

MP4-1 showed more effective blocking-activity than PF2566.

[0088] Example 4. MP4-1 and 2D10 inhibited tumor growth

[0089] Isolation ofPBMCs and T cells

[0090] Freshly prepared whole blood was obtained from healthy donors and human peripheral blood mononuclear cells (PBMCs) isolated using Ficoll (GE Healthcare) density gradient centrifugation according to the instruction. The EasySep Human CD3

Positive Selection Kit (STEMCELL Technologies Inc., Vancouver, Canada) was used to

purify T cells. Isolated T cells were cultured in completed RPMI 1640 with 10% fetal

bovine serum and 1% Penicillin/Streptomycin at 37°C in a 5 % C02 humidified

incubator before assays.

[0091] Xenograft models

[0092] Four to five weeks old NOD/SCID mice were injected with the cell mixtures of ix106 LS174T cells and 5x106 human PBMCs in 200ul PBS at the right flank. Two

hours after the engraftment, 4-1BB antibody (5mg/kg) or vehicle control (PBS) were

administered intra-peritoneal. The animals were then treated (100pg per mouse) on days

0, 2, 6, 8, 10 and 12. Tumor volume was measured with calipers in 2 perpendicular 2 dimensions and was calculated using the formula width x length)/2.

[0093] Result: To further investigate whether 4-1BB antibody could inhibit tumor cell

growth in vivo, MP4-1 and 2D10 were used to evaluate anti-tumor activity in

NOD/SCID mice. As shown in Figure 4, from day 10, the tumor volume of each group

started to increase, and the tumor volume of the vehicle group increased the fastest,

which was significantly faster than that of the 4-1BB MP4-1 group and the 4-1BB 2D10

group. On day 14, the tumor volume of 4-1BB MP4-1 group and 4-1BB 2D10 group

was not significantly different, but both were significantly smaller (P<0.001) than the

tumor volume of vehicle group. These data demonstrate that 4-1BB antibodies MP4-1

and 2D10 can effectively inhibit tumor growth in a xenograft mouse model.

[0094] Example 5. Characterization of humanized MP4-1

[0095] Based on the binding affinity and the activity of 4-1BB antibodies to block the

interaction of 4-1BB and its ligand, we chose MP4-1 for further humanization. 9

candidates with human IgGI structure were constructed and tested 4-BB binding

affinity. As shown in Figure 5A, only PP9150 and PP9153 have KD value at the

nanomolar scale. However, only PP9150 showed similar blocking activity to MP4-1 in

antibody 4-1BB ligand competition assay (Figure 5B). We then tested the binding of

PP9150 to cell membrane bound 4-1BB, same as the control PF2566, no binding of

PP9150 to 4-1BB negative Jurkat cells were observed (Figure 6A). Specific binding of

PP9150 and PF2566 to the activated T cells was detected (Figure 6B).

[0096] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the

common general knowledge.

[0097] It will be understood that the terms "comprise" and "include" and any of their derivatives (e.g. comprises, comprising, includes, including) as used in this specification,

and the claims that follow, is to be taken to be inclusive of features to which the term

refers, and is not meant to exclude the presence of any additional features unless otherwise

stated or implied.

SEQUENCE LISTING SEQUENCE LISTING

<110> SOUND BIOPHARMACEUTICALS <110> SOUND BIOPHARMACEUTICALSCO. CO.LTD. LTD.

<120> Humanized4-1BB <120> Humanized 4-1BBmonoclonal monoclonalantibody antibodyand andpharmaceutical pharmaceuticalcomposition composition thereof thereof

<130> <130> 193405-22D-AUP 193405-22D-AUP

<150> <150> 201911133718.7 201911133718.7 <151> <151> 2019-11-19 2019-11-19

<160> <160> 16 16

<170> PatentInversion <170> PatentIn version3.5 3.5

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Trp Ile Trp Ile Ser Ser Trp Trp Val Val Arg Arg 1 1 5 5

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Val Val

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<220> <220>

<223> Heavychain <223> Heavy chaincomplementarity complementaritydetermining determiningregion regionCDR3 CDR3of ofhumanized humanized4-1BB 4-1BB

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Cys Ala Cys Ala Arg ArgArg ArgThr Thr ArgArg TyrTyr Asp Asp Tyr Tyr Glu Tyr Glu Asp Asp Phe TyrAla PheMet Ala AspMet Asp 1 1 5 5 10 10 15 15

<210> <210> 4 4 <211> <211> 6 6 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

<220> <220> <223> Heavy chain <223> Heavy chain complementarity complementarity determining determining region region CDR1 CDR1 of of humanized humanized 4-1BB 4-1BB

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Val Val

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<220> <220> <223> Light chain <223> Light chain complementarity complementarity determining determining region region CDR1 CDR1'ofofhumanized humanized4-1BB 4-1BB

<400> <400> 6 6

Cys Gln Cys Gln Ala AlaSer SerGln Gln GlyGly IleIle Asn Asn Gln Gln Tyr Tyr Leu Leu 1 1 5 5 10

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<400> <400> 8 8

Ile Ala Thr Ile Ala ThrTyr TyrTyr Tyr CysCys GlnGln Gln Gln Tyr Tyr Ser Ser Glu Pro Glu Leu LeuPhe Pro Phe 1 1 5 5 10 10

<210> <210> 9 9 <211> <211> 221 221 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

<220> <220> <223> Theheavy <223> The heavy chain chain variable variable region region VH of VH of humanized humanized 4-1BB, 4-1BB, - Xaa isXaa Glu is or Glu Gln or Gln

<400> <400> 9 9

Xaa Val Xaa Val Gln GlnLeu LeuVal Val GlnGln SerSer Gly Gly Ala Ala Glu Lys Glu Val Val Lys LysPro LysGly Pro SerGly Ser 1 1 5 5 10 10 15 15

Ser Val Lys Ser Val LysVal ValSer Ser CysCys LysLys Ala Ala Ser Ser Gly Gly Tyr Phe Tyr Gly GlySer PheSer SerSerSer Ser 20 20 25 25 30 30

Trp Ile Trp Ile Ser SerTrp TrpVal Val ArgArg GlnGln Ala Ala Pro Pro Gly Gly Gly Gln Gln Leu GlyGlu LeuTrp Glu MetTrp Met 35 35 40 40 45 45

Gly Arg Gly Arg Ile IleHis HisPro Pro GlyGly GluGlu Gly Gly Glu Glu Thr Tyr Thr Tyr Tyr Ala TyrGln AlaLys Gln PheLys Phe 50 50 55 55 60

Gln Gly Gln Gly Arg ArgVal ValThr Thr IleIle ThrThr Ala Ala Asp Asp Lys Thr Lys Ser Ser Ser ThrThr SerAla Thr TyrAla Tyr

70 70 75 75 80 80

Met Glu Met Glu Leu LeuSer SerSer Ser LeuLeu ArgArg Ser Ser Glu Glu Asp Ala Asp Thr Thr Val AlaTyr ValTyr Tyr CysTyr Cys 85 85 90 90 95 95

Ala Arg Ala Arg Arg ArgThr ThrArg Arg TyrTyr AspAsp Tyr Tyr Glu Glu Asp Phe Asp Tyr Tyr Ala PheMet AlaAsp Met TyrAsp Tyr 100 100 105 105 110 110

Trp Gly Trp Gly Gln Gln Gly Gly Thr Thr Leu Leu Val Val Thr Thr Val Val Ser Ser Ser Ser Ala Ala Ser Ser Thr Thr Lys Lys Gly Gly 115 115 120 120 125 125

Pro Ser Pro Ser Val ValPhe PhePro Pro LeuLeu AlaAla Pro Pro Ser Ser Ser Ser Ser Lys Lys Thr SerSer ThrGly Ser GlyGly Gly 130 130 135 135 140 140

Thr Ala Thr Ala Ala AlaLeu LeuGly Gly CysCys LeuLeu Val Val Lys Lys Asp Phe Asp Tyr Tyr Pro PheGlu ProPro Glu ValPro Val 145 145 150 150 155 155 160 160

Thr Val Thr Val Ser SerTrp TrpAsn Asn SerSer GlyGly Ala Ala Leu Leu Thr Gly Thr Ser Ser Val GlyHis ValThr His PheThr Phe 165 165 170 170 175 175

Pro Ala Val Pro Ala ValLeu LeuGln Gln SerSer SerSer Gly Gly Leu Leu Tyr Leu Tyr Ser Ser Ser LeuSer SerVal Ser ValVal Val 180 180 185 185 190 190

Thr Val Thr Val Pro ProSer SerSer Ser SerSer LeuLeu Gly Gly Thr Thr Gln Tyr Gln Thr Thr Ile TyrCys IleAsn Cys ValAsn Val 195 195 200 200 205 205

Asn His Asn His Lys LysPro ProSer Ser AsnAsn ThrThr Lys Lys Val Val Asp Arg Asp Lys Lys Val Arg Val 210 210 215 215 220 220

<210> <210> 10 10 <211> <211> 221 221 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

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<400> <400> 10 10

Glu Val Glu Val Gln GlnLeu LeuVal Val GluGlu SerSer Gly Gly Ala Ala Glu Lys Glu Val Val Lys LysPro LysGly Pro AlaGly Ala 1 1 5 5 10 10 15

Ser Val Ser Val Lys LysVal ValSer Ser CysCys LysLys Ala Ala Ser Ser Gly Gly Gly Tyr Tyr Phe GlySer PheSer SerSerSer Ser 20 20 25 25 30 30

Trp Met Trp Met Asn AsnTrp TrpVal Val ArgArg GlnGln Ala Ala Pro Pro Gly Gly Gly Gln Gln Leu GlyGlu LeuTrp Glu MetTrp Met 35 35 40 40 45 45

Gly Arg Gly Arg Ile IleHis HisPro Pro GlyGly AspAsp Gly Gly Glu Glu Thr Tyr Thr Tyr Tyr Asn TyrGln AsnLys Gln PheLys Phe 50 50 55 55 60 60

Gln Gly Gln Gly Arg ArgVal ValThr Thr MetMet ThrThr Ala Ala Asp Asp Lys Thr Lys Ser Ser Ser ThrThr SerVal Thr TyrVal Tyr

70 70 75 75 80 80

Trp Gly Trp Gly Gln GlnGly GlyThr Thr ThrThr ValVal Thr Thr Val Val Ser Ala Ser Ser Ser Ser AlaThr SerLys Thr GlyLys Gly 115 115 120 120 125 125

Pro Ser Val Pro Ser ValPhe PhePro Pro LeuLeu AlaAla Pro Pro Cys Cys Ser Ser Ser Arg Arg Thr SerSer ThrGlu Ser SerGlu Ser 130 130 135 135 140 140

Pro Ala Pro Ala Val ValLeu LeuGln Gln SerSer SerSer Gly Gly Leu Leu Tyr Leu Tyr Ser Ser Ser LeuSer SerVal Ser ValVal Val 180 180 185 185 190 190

Thr Val Thr Val Pro ProSer SerSer Ser AsnAsn PhePhe Gly Gly Thr Thr Gln Tyr Gln Thr Thr Thr TyrCys ThrAsn Cys ValAsn Val 195 195 200 200 205 205

Asp His Asp His Lys LysPro ProSer Ser AsnAsn ThrThr Lys Lys Val Val Asp Thr Asp Lys Lys Val Thr Val 210 210 215 215 220 220

<210> <210> 11 11 <211> <211> 214

<212> PRT <212> PRT <213> <213> Artificial Sequence Artificial Sequence

<220> <220> <223> Light chain <223> Light chain variable variable region region VL VL of of humanized humanized 4-1BB 4-1BB

<400> <400> 11 11

Asp Ile Asp Ile Gln Gln Met Met Thr Thr Gln Gln Ser Ser Pro Pro Ser Ser Ser Ser Leu Leu Ser Ser Ala Ala Ser Ser Val Val Gly Gly 1 1 5 5 10 10 15 15

Asp Arg Asp Arg Val ValThr ThrIle Ile ThrThr CysCys Gln Gln Ala Ala Ser Gly Ser Gln Gln Ile GlyAsn IleGln AsnTyrGln Tyr 20 20 25 25 30 30

Leu Asn Trp Leu Asn TrpTyr TyrGln Gln Gln Gln LysLys ProPro Gly Gly Lys Lys Ala Lys Ala Pro ProLeu LysLeu Leu Leu Ile Ile 35 35 40 40 45 45

Phe Tyr Thr Phe Tyr ThrSer SerSer Ser LeuLeu HisHis Thr Thr Gly Gly Val Val Pro Arg Pro Ser SerPhe ArgSer Phe GlySer Gly 50 50 55 55 60 60

Ser Gly Ser Ser Gly SerGly GlyThr Thr AspAsp TyrTyr Thr Thr Phe Phe Thr Ser Thr Ile Ile Ser SerLeu SerGln Leu ProGln Pro

70 70 75 75 80 80

Glu Asp Glu Asp Ile IleAla AlaThr ThrTyrTyr TyrTyr Cys Cys Gln Gln Gln Ser Gln Tyr Tyr Glu SerLeu GluPro Leu PhePro Phe 85 85 90 90 95 95

Thr Phe Thr Phe Gly GlyGln GlnGly Gly ThrThr LysLys Leu Leu Glu Glu Ile Arg Ile Lys Lys Thr ArgVal ThrAla Val AlaAla Ala 100 100 105 105 110 110

Pro Ser Val Pro Ser ValPhe PheIle Ile PhePhe ProPro Pro Pro Ser Ser Asp Asp Glu Leu Glu Gln GlnLys LeuSer Lys GlySer Gly 115 115 120 120 125 125

Thr Ala Thr Ala Ser SerVal ValVal Val CysCys LeuLeu Leu Leu Asn Asn Asn Tyr Asn Phe Phe Pro TyrArg ProGlu Arg AlaGlu Ala 130 130 135 135 140 140

Lys Val Gln Lys Val GlnTrp TrpLys Lys Val Val AspAsp AsnAsn Ala Ala Leu Leu Gln Gly Gln Ser SerAsn GlySer Asn Ser Gln Gln 145 145 150 150 155 155 160 160

Glu Ser Glu Ser Val ValThr ThrGlu Glu GlnGln AspAsp Ser Ser Lys Lys Asp Thr Asp Ser Ser Tyr ThrSer TyrLeu Ser SerLeu Ser 165 165 170 170 175 175

Ser Thr Leu Ser Thr LeuThr ThrLeu Leu SerSer LysLys Ala Ala Asp Asp Tyr Lys Tyr Glu Glu His LysLys HisVal Lys TyrVal Tyr 180 180 185 185 190

Ala Cys Ala Cys Glu GluVal ValThr Thr HisHis GlnGln Gly Gly Leu Leu Ser Pro Ser Ser Ser Val ProThr ValLys Thr SerLys Ser 195 195 200 200 205 205

Phe Asn Arg Phe Asn ArgGly GlyGlu Glu CysCys 210 210

<210> <210> 12 12 <211> <211> 472 472 <212> <212> PRT PRT <213> <213> ArtificialSequence Artificial Sequence

<220> <220> <223> Theheavy <223> The heavy chain chain of humanized of humanized 4-1BB, 4 1-1BB, Xaais is - Xaa GluGlu or or Gln Gln

<400> <400> 12 12

Met Asp Met Asp Pro ProLys LysGly Gly SerSer LeuLeu Ser Ser Trp Trp Arg Leu Arg Ile Ile Leu LeuPhe LeuLeu Phe SerLeu Ser 1 1 5 5 10 10 15 15

Leu Ala Phe Leu Ala PheGlu GluLeu Leu Ser Ser TyrTyr Gly Gly Xaa Xaa Val Val Gln Val Gln Leu LeuGln ValSer GlnGlySer Gly 20 20 25 25 30 30

Ala Glu Ala Glu Val ValLys LysLys Lys ProPro GlyGly Ser Ser Ser Ser Val Val Val Lys Lys Ser ValCys SerLys Cys AlaLys Ala 35 35 40 40 45 45

Ser Gly Tyr Ser Gly TyrGly GlyPhe Phe SerSer SerSer Ser Ser Trp Trp Ile Trp Ile Asn Asn Val TrpArg ValGln Arg AlaGln Ala 50 50 55 55 60 60

Pro Gly Gln Pro Gly GlnGly GlyLeu Leu GluGlu TrpTrp Met Met Gly Gly Arg His Arg Ile Ile Pro HisGly ProAsp Gly GlyAsp Gly

70 70 75 75 80 80

Glu Thr Glu Thr Tyr TyrTyr TyrAla AlaGlnGln LysLys Phe Phe Gln Gln Gly Val Gly Arg Arg Thr ValIle ThrThr Ile AlaThr Ala 85 85 90 90 95 95

Asp Lys Asp Lys Ser SerThr ThrSer Ser ThrThr AlaAla Tyr Tyr Met Met Glu Ser Glu Leu Leu Ser SerLeu SerArg Leu SerArg Ser 100 100 105 105 110 110

Glu Asp Glu Asp Thr ThrAla AlaVal Val TyrTyr TyrTyr Cys Cys Ala Ala Arg Thr Arg Arg Arg Arg ThrTyr ArgAsp Tyr TyrAsp Tyr 115 115 120 120 125 125

Glu Asp Glu Asp Tyr TyrPhe PheAla Ala MetMet AspAsp Tyr Tyr Trp Trp Gly Gly Gly Gln Gln Thr GlyLeu ThrVal Leu ThrVal Thr

130 135 135 140 140

Val Ser Val Ser Ser Ser Ala Ala Ser Ser Thr Thr Lys Lys Gly Gly Pro Pro Ser Ser Val Val Phe Phe Pro Pro Leu Leu Ala Ala Pro Pro 145 145 150 150 155 155 160 160

Cys Ser Cys Ser Arg ArgSer SerThr Thr SerSer GluGlu Ser Ser Thr Thr Ala Leu Ala Ala Ala Gly LeuCys GlyLeu Cys ValLeu Val 165 165 170 170 175 175

Lys Asp Tyr Lys Asp TyrPhe PhePro Pro Glu Glu ProPro ValVal Thr Thr Val Val Ser Asn Ser Trp TrpSer AsnGly Ser Gly Ala Ala 180 180 185 185 190 190

Leu Thr Ser Leu Thr SerGly GlyVal Val His His ThrThr Phe Phe Pro Pro Ala Ala Val Gln Val Leu LeuSer GlnSer Ser GlySer Gly 195 195 200 200 205 205

Leu Tyr Ser Leu Tyr SerLeu LeuSer Ser SerSer ValVal Val Val Thr Thr Val Val Pro Ser Pro Ser SerAsn SerPhe Asn GlyPhe Gly 210 210 215 215 220 220

Thr Gln Thr Gln Thr ThrTyr TyrThr Thr CysCys AsnAsn Val Val Asp Asp His Pro His Lys Lys Ser ProAsn SerThr Asn LysThr Lys 225 225 230 230 235 235 240 240

Val Asp Val Asp Lys LysThr ThrVal Val GluGlu ArgArg Lys Lys Cys Cys Cys Glu Cys Val Val Cys GluPro CysPro Pro CysPro Cys 245 245 250 250 255 255

Pro Ala Pro Ala Pro ProPro ProVal Val AlaAla GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro PhePro Pro LysPro Lys 260 260 265 265 270 270

Pro Lys Pro Lys Asp AspThr ThrLeu Leu MetMet IleIle Ser Ser Arg Arg Thr Glu Thr Pro Pro Val GluThr ValCys Thr ValCys Val 275 275 280 280 285 285

Val Val Val Val Asp Asp Val Val Ser Ser His His Glu Glu Asp Asp Pro Pro Glu Glu Val Val Gln Gln Phe Phe Asn Asn Trp Trp Tyr Tyr 290 290 295 295 300 300

Val Asp Val Asp Gly GlyVal ValGlu Glu ValVal HisHis Asn Asn Ala Ala Lys Lys Lys Thr Thr Pro LysArg ProGlu Arg GluGlu Glu 305 305 310 310 315 315 320 320

Gln Phe Gln Phe Asn AsnSer SerThr Thr PhePhe ArgArg Val Val Val Val Ser Leu Ser Val Val Thr LeuVal ThrVal Val HisVal His 325 325 330 330 335 335

Gln Asp Gln Asp Trp TrpLeu LeuAsn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer ValAsn Ser LysAsn Lys 340 340 345 345 350

Gly Leu Gly Leu Pro ProAla AlaPro Pro IleIle GluGlu Lys Lys Thr Thr Ile Lys Ile Ser Ser Thr LysLys ThrGly Lys GlnGly Gln 355 355 360 360 365 365

Pro Arg Glu Pro Arg GluPro ProGln Gln ValVal TyrTyr Thr Thr Leu Leu Pro Ser Pro Pro Pro Arg SerGlu ArgGlu Glu MetGlu Met 370 370 375 375 380 380

Thr Lys Thr Lys Asn Asn Gln Gln Val Val Ser Ser Leu Leu Thr Thr Cys Cys Leu Leu Val Val Lys Lys Gly Gly Phe Phe Tyr Tyr Pro Pro 385 385 390 390 395 395 400 400

Ser Asp Ile Ser Asp IleAla AlaVal Val GluGlu TrpTrp Glu Glu Ser Ser Asn Gln Asn Gly Gly Pro GlnGlu ProAsn Glu AsnAsn Asn 405 405 410 410 415 415

Tyr Lys Tyr Lys Thr ThrThr ThrPro Pro ProPro MetMet Leu Leu Asp Asp Ser Gly Ser Asp Asp Ser GlyPhe SerPhe Phe LeuPhe Leu 420 420 425 425 430 430

Tyr Ser Tyr Ser Lys LysLeu LeuThr Thr ValVal AspAsp Lys Lys Ser Ser Arg Gln Arg Trp Trp Gln GlnGly GlnAsn Gly ValAsn Val 435 435 440 440 445 445

Phe Ser Cys Phe Ser CysSer SerVal Val MetMet HisHis Glu Glu Ala Ala Leu Asn Leu His His His AsnTyr HisThr Tyr GlnThr Gln 450 450 455 455 460 460

Lys Ser Leu Lys Ser LeuSer SerLeu Leu Ser Ser ProPro GlyGly 465 465 470 470

<210> <210> 13 13 <211> <211> 472 472 <212> <212> PRT PRT <213> <213> ArtificialSequence Artificial Sequence

<220> <220> <223> The heavy <223> The heavy chain chain of of humanized humanized 4-1BB 4-1BB

<400> <400> 13 13

Met Asp Met Asp Pro Pro Lys Lys Gly Gly Ser Ser Leu Leu Ser Ser Trp Trp Arg Arg Ile Ile Leu Leu Leu Leu Phe Phe Leu Leu Ser Ser 1 1 5 5 10 10 15 15

Leu Ala Phe Leu Ala PheGlu GluLeu Leu SerSer TyrTyr Gly Gly Glu Glu Val Val Gln Val Gln Leu LeuGlu ValSer GluGlySer Gly 20 20 25 25 30 30

Ala Glu Ala Glu Val Val Lys Lys Lys Lys Pro Pro Gly Gly Ala Ala Ser Ser Val Val Lys Lys Val Val Ser Ser Cys Cys Lys Lys Ala Ala 35 35 40 40 45

Ser Gly Tyr Ser Gly TyrGly GlyPhe Phe SerSer SerSer Ser Ser Trp Trp Met Trp Met Asn Asn Val TrpArg ValGln Arg AlaGln Ala 50 50 55 55 60 60

Pro Gly Gln Pro Gly GlnGly GlyLeu Leu GluGlu TrpTrp Met Met Gly Gly Arg Arg Ile Pro Ile His HisGly ProAsp Gly GlyAsp Gly

70 70 75 75 80 80

Glu Thr Glu Thr Tyr TyrTyr TyrAsn Asn GlnGln LysLys Phe Phe Gln Gln Gly Val Gly Arg Arg Thr ValMet ThrThr Met AlaThr Ala 85 85 90 90 95 95

Asp Lys Asp Lys Ser Ser Thr Thr Ser Ser Thr Thr Val Val Tyr Tyr Met Met Glu Glu Leu Leu Ser Ser Ser Ser Leu Leu Arg Arg Ser Ser 100 100 105 105 110 110

Glu Asp Glu Asp Tyr TyrPhe PheAla Ala MetMet AspAsp Tyr Tyr Trp Trp Gly Gly Gly Gln Gln Thr GlyThr ThrVal Thr ThrVal Thr 130 130 135 135 140 140

Lys Asp Tyr Lys Asp TyrPhe PhePro Pro Glu Glu ProPro ValVal Thr Thr Val Val Ser Asn Ser Trp TrpSer AsnGly Ser AlaGly Ala 180 180 185 185 190 190

Leu Thr Ser Leu Thr SerGly GlyVal Val His His ThrThr PhePhe Pro Pro Ala Ala Val Gln Val Leu LeuSer GlnSer Ser GlySer Gly 195 195 200 200 205 205

Leu Tyr Ser Leu Tyr SerLeu LeuSer Ser Ser Ser ValVal Val Val Thr Thr Val Val Pro Ser Pro Ser SerAsn SerPhe Asn GlyPhe Gly 210 210 215 215 220 220

Pro Ala Pro Ala Pro ProPro ProVal Val AlaAla GlyGly Pro Pro Ser Ser Val Leu Val Phe Phe Phe LeuPro PhePro Pro LysPro Lys

260 265 265 270 270

Pro Lys Asp Pro Lys AspThr ThrLeu Leu MetMet IleIle Ser Ser Arg Arg Thr Thr Pro Val Pro Glu GluThr ValCys Thr ValCys Val 275 275 280 280 285 285

Val Asp Val Asp Gly Gly Val Val Glu Glu Val Val His His Asn Asn Ala Ala Lys Lys Thr Thr Lys Lys Pro Pro Arg Arg Glu Glu Glu Glu 305 305 310 310 315 315 320 320

Gln Asp Gln Asp Trp TrpLeu LeuAsn Asn GlyGly LysLys Glu Glu Tyr Tyr Lys Lys Lys Cys Cys Val LysSer ValAsn Ser LysAsn Lys 340 340 345 345 350 350

Pro Arg Glu Pro Arg GluPro ProGln Gln ValVal TyrTyr Thr Thr Leu Leu Pro Pro Pro Arg Pro Ser SerGlu ArgGlu Glu MetGlu Met 370 370 375 375 380 380

Thr Lys Thr Lys Asn AsnGln GlnVal Val SerSer LeuLeu Thr Thr Cys Cys Leu Lys Leu Val Val Gly LysPhe GlyTyr Phe ProTyr Pro 385 385 390 390 395 395 400 400

Lys Ser Leu Lys Ser LeuSer SerLeu Leu Ser Ser ProPro GlyGly 465 465 470

<210> <210> 14 14 <211> <211> 234 234 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

<220> <220> <223> <223> The lightchain The light chainofofhumanized humanized 4-1BB 4-1BB

<400> <400> 14 14

Met Glu Met Glu Thr ThrAsp AspThr Thr LeuLeu LeuLeu Leu Leu Trp Trp Val Leu Val Leu Leu Leu LeuTrp LeuVal Trp ProVal Pro 1 1 5 5 10 10 15 15

Gly Ser Gly Ser Thr ThrGly GlyAsp Asp IleIle GlnGln Met Met Thr Thr Gln Pro Gln Ser Ser Ser ProSer SerLeu SerSerLeu Ser 20 20 25 25 30 30

Ala Ser Ala Ser Val Val Gly Gly Asp Asp Arg Arg Val Val Thr Thr Ile Ile Thr Thr Cys Cys Gln Gln Ala Ala Ser Ser Gln Gln Gly Gly 35 35 40 40 45 45

Ile Asn Gln Ile Asn GlnTyr TyrLeu Leu AsnAsn TrpTrp Tyr Tyr Gln Gln Gln Gln Lys Gly Lys Pro ProLys GlyAla Lys ProAla Pro 50 50 55 55 60 60

Lys Leu Leu Lys Leu LeuIle IlePhe Phe Tyr Tyr ThrThr SerSer Ser Ser Leu Leu His Gly His Thr ThrVal GlyPro Val Pro Ser Ser

70 70 75 75 80 80

Arg Phe Arg Phe Ser SerGly GlySer SerGlyGly SerSer Gly Gly Thr Thr Asp Thr Asp Tyr Tyr Phe ThrThr PheIle Thr SerIle Ser 85 85 90 90 95 95

Ser Leu Gln Ser Leu GlnPro ProGlu Glu AspAsp IleIle Ala Ala Thr Thr Tyr Cys Tyr Tyr Tyr Gln CysGln GlnTyr Gln SerTyr Ser 100 100 105 105 110 110

Glu Leu Glu Leu Pro Pro Phe Phe Thr Thr Phe Phe Gly Gly Gln Gln Gly Gly Thr Thr Lys Lys Leu Leu Glu Glu Ile Ile Lys Lys Arg Arg 115 115 120 120 125 125

Thr Val Thr Val Ala AlaAla AlaPro Pro SerSer ValVal Phe Phe Ile Ile Phe Pro Phe Pro Pro Ser ProAsp SerGlu Asp GlnGlu Gln 130 130 135 135 140 140

Leu Lys Ser Leu Lys SerGly GlyThr Thr Ala Ala SerSer ValVal Val Val Cys Cys Leu Asn Leu Leu LeuAsn AsnPhe Asn Phe Tyr Tyr 145 145 150 150 155 155 160 160

Pro Arg Glu Pro Arg GluAla AlaLys Lys ValVal GlnGln Trp Trp Lys Lys Val Val Asp Ala Asp Asn AsnLeu AlaGln Leu SerGln Ser 165 165 170 170 175

Gly Asn Gly Asn Ser SerGln GlnGlu Glu SerSer ValVal Thr Thr Glu Glu Gln Ser Gln Asp Asp Lys SerAsp LysSer Asp ThrSer Thr 180 180 185 185 190 190

Tyr Ser Tyr Ser Leu LeuSer SerSer Ser ThrThr LeuLeu Thr Thr Leu Leu Ser Ala Ser Lys Lys Asp AlaTyr AspGlu Tyr LysGlu Lys 195 195 200 200 205 205

His Lys Val His Lys ValTyr TyrAla Ala CysCys GluGlu Val Val Thr Thr His Gly His Gln Gln Leu GlySer LeuSer Ser ProSer Pro 210 210 215 215 220 220

Val Thr Val Thr Lys LysSer SerPhe Phe AsnAsn ArgArg Gly Gly Glu Glu Cys Cys 225 225 230 230

<210> <210> 15 15 <211> <211> 24 24 <212> <212> PRT PRT <213> <213> Artificial Sequence Artificial Sequence

<220> <220> <223> <223> signal peptide signal peptide

<400> <400> 15 15

Leu Ala Phe Leu Ala PheGlu GluLeu Leu Ser Ser TyrTyr GlyGly 20 20

<210> <210> 16 16 <211> <211> 20 20 <212> <212> PRT PRT <213> <213> ArtificialSequence Artificial Sequence

<220> <220> <223> <223> signal peptide signal peptide

<400> <400> 16 16

Gly Ser Gly Ser Thr ThrGly Gly 20

Claims

The claims defining the invention are as follows:

1. A humanized 4-1BB monoclonal antibody or antigen-binding fragment thereof,

wherein a heavy chain variable region of the humanized 4-1BB monoclonal antibody

is the heavy chain variable region contained in the amino acid sequence shown in SEQ

ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12, and a light chain variable region of the

humanized 4-1BB monoclonal antibody is the light chain variable region contained in

the amino acid sequence shown in SEQ ID NO: 11.

2. The humanized 4-1BB monoclonal antibody or antigen-binding fragment thereof of

claim 1, wherein a heavy chain of the humanized 4-1BB monoclonal antibody is the

amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO: 13, and a light chain of

the humanized 4-1BB monoclonal antibody is the amino acid sequence shown in SEQ

ID NO: 14.

3. The humanized 4-1BB monoclonal antibody or antigen-binding fragment thereof of

claim 1, wherein the antigen-binding fragment is selected from scFv, (scFv)2, Fab, Fab'

and F(ab')2 of the humanized 4-1BB monoclonal antibody.

4. A pharmaceutical composition comprising the humanized 4-1BB monoclonal

antibody or antigen-binding fragment thereof of any one of claims 1-3 and a

pharmaceutically acceptable carrier.

5. A pharmaceutical composition comprising the humanized 4-1BB monoclonal

antibody or antigen-binding fragment thereof of any one of claims 1-3 and a second

anti-cancer agent.

6. The pharmaceutical composition of claim 5, wherein the second anti-cancer agent is

selected from PD-1 antibody, CTLA-4 antibody, GM-CSF, CD40 antibody, cisplatin,

cyclophosphamide, cytokine-induced killer cells and IL-2.

7. Use of the humanized 4-1BB monoclonal antibody or antigen-binding fragment

thereof of any one of claims 1-3 or the pharmaceutical composition of any one of claims

4-6 in the manufacture of a medicament for treating cancer, autoimmune diseases,

inflammatory diseases, viral infections or a disease associated with a viral infection.

8. The use of claim 7, wherein the cancer is selected from esophageal cancer, stomach

cancer, colon cancer, rectal cancer, pancreatic cancer, lung cancer, breast cancer,

cervical cancer, corpus carcinoma, ovarian cancer, bladder cancer, head and neck cancer,

endometrial cancer, osteosarcoma, prostate cancer, and neuroblastoma.

9. The use of claim 7, wherein the autoimmune disease is selected from Graves'disease,

multiple sclerosis, autoimmune liver disease, primary adrenal atrophy, chronic

thyroiditis, type 1 diabetes, systemic lupus erythematosus, psoriasis, Crohn's disease,

atopic dermatitis, autoimmune hemolytic anemia, myasthenia gravis, demyelinating

disease, eczema, graft-versus-host disease, rheumatoid arthritis, scleroderma, sjogren

syndrome, chronic nephritis, ankylosing spondylitis, chronic active hepatitis, atrophic

gastritis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome,

idiopathic thrombocytopenic purpura, idiopathic leukopenia, chronic thyroiditis,

pernicious anemia, chronic ulcerative colitis.

10. The use of claim 7, wherein the inflammatory disease is selected from arthritis,

tendinitis, arteriosclerosis, polymyalgia rheumatica, bursitis, cystic fibrosis, arthrosteitis, giant cell arteritis, polymyositis, dermatomyositis, pemphigus, pemphigoid, mixed connective tissue disease, sclerosing cholangitis, inflammatory

bowel disease, ulcerative colitis, inflammatory skin diseases, asbestosis, silicosis,

pneumoconiosis, sarcoidosis, extrinsic allergic alveolitis, hepatitis, delayed type

hypersensitivity, pneumonia, respiratory tract inflammation, adult respiratory distress

syndrome, encephalitis, immediate hypersensitivity, asthma, hay fever, allergies, acute

anaphylaxis, rheumatic fever, cystitis, chronic cholecystitis, allograft rejection, host

versus-graft rejection, appendicitis, arteritis, bronchiolitis, bronchitis, cervicitis,

cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis,

endocarditis, endometritis, enteritis, epicondylitis, epididymitis, fasciitis, fibrositis,

gastritis, gastroenteritis, gingivitis, iritis, laryngitis, myelitis, myocarditis, nephritis,

omphalitis, oophoritis, orchitis, osteitis, otitis, pancreatitis, mumps, pericarditis,

pharyngitis, pleurisy, phlebitis, prostatitis, rhinitis, salpingitis, sinusitis, stomatitis,

synovitis, orchitis, tonsillitis, urethritis, uveitis, vaginitis, vulvitis, vasculitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis, and cardiovascular inflammation.

11. The use of claim 7, wherein the disease associated with the viral infection is selected

from retinitis, enteritis, infectious mononucleosis, Hodgkin's lymphoma, Burkitt's

lymphoma, nasopharyngeal carcinoma, acquired immune deficiency syndrome, upper

respiratory tract infection, lower respiratory tract infection, myocarditis, encephalitis,

dengue haemorrhagic fever/dengue shock syndrome, hepatitis A, hepatitis B, hepatitis

C, gingivostomatitis, keratoconjunctivitis, skin papules, mumps, polio, rabies, rubella

and chickenpox.

12. A nucleotide sequence encoding the humanized 4-1BB monoclonal antibody or

antigen-binding fragment thereof of any one of claims 1-3.

13. A vector comprising the nucleotide sequence of claim 12.

14. A non-human host cell comprising the vector of claim 13.

15. A method of treating cancer, autoimmune diseases, inflammatory diseases, viral

infections or a disease associated with a viral infection in a subject, comprising

administering the humanized 4-1BB monoclonal antibody or antigen-binding fragment

4-6 to the subject.

16. The method of claim 15, wherein the cancer is selected from esophageal cancer,

17. The method of claim 15, wherein the autoimmune disease is selected from Graves'

disease, multiple sclerosis, autoimmune liver disease, primary adrenal atrophy, chronic

syndrome, chronic nephritis, ankylosing spondylitis, chronic active hepatitis, atrophic gastritis, autoimmune glomerulonephritis, pulmonary and renal hemorrhagic syndrome, idiopathic thrombocytopenic purpura, idiopathic leukopenia, chronic thyroiditis, pernicious anemia, chronic ulcerative colitis.

18. The method of claim 15, wherein the inflammatory disease is selected from arthritis,

cholangitis, chorioamnionitis, conjunctivitis, dacryoadenitis, dermatomyositis,

synovitis, orchitis, tonsillitis, urethritis, uveitis, vaginitis, vulvitis, vasculitis, osteomyelitis, optic neuritis, temporal arteritis, transverse myelitis, necrotizing fasciitis,

and cardiovascular inflammation.

19. The method of claim 15, wherein the disease associated with the viral infection is

selected from retinitis, enteritis, infectious mononucleosis, Hodgkin's lymphoma,

Burkitt's lymphoma, nasopharyngeal carcinoma, acquired immune deficiency

syndrome, upper respiratory tract infection, lower respiratory tract infection,

myocarditis, encephalitis, dengue haemorrhagic fever/dengue shock syndrome,

hepatitis A, hepatitis B, hepatitis C, gingivostomatitis, keratoconjunctivitis, skin

papules, mumps, polio, rabies, rubella and chickenpox.

Figures Figures

1.5i 1.5 —Animal - Animal11 A B B A —Animal - Animal22 £ aCM 1.0" 1.0 —Animal - Animal33 IO Antigen Antigen —Animal - Animal44 BB lymphocytes lymphocytes SP2/0cells SP2/0 cells E Spleen Spleen o Fusion Fusion o 5 |0 — Blank - Blankserum serum "V — 0.5 0.5- Q HATselection HAT selection O

o o Hybridcells cells o 0.0' 0.0 Hybrid ^ ^ ^ ^ ^ ^ ^ Singlecell Single cell selection selection K nN ^ # v < ^ ELISAselection ELISA selection \ N- Hybridoma Hybridoma cells cells Dilutionratio Dilution ratio Hybridoma AA Q Hybridoma f~_") Hybridoma CC Hybridoma c C Clone Clone Hybridoma BB Hybridoma Label Label ka (1/Ms) ka (1/Ms) kU (1/s) kd (1/s) Kd (nM) KD (nM) Number I MP4-1 MP4-1 FPI-1 FP1-1 Number 6C1A8 6.66E+05 6C1A8 2D10 6.66E+05 4.90E+05 4.90E+05 5.76E-05 5.76E-05

3.50E-03 3.50E-03 0.0865 0.0865

7.14 7.14 Ascites Ascites ProteinAApurification Protein purification 2D10 FPl-3 FP1-3 9B4 9B4 3.88E+05 3.88E+05 3.62E-03 3.62E-03 9.34 9.34 FP2-12 FP2-12 1G4 1G4 8.08E+05 8.08E+05 2.49E-03 2.49E-03 3.09 3.09 FP2-14 FP2-14 10 A3 10A3 5.63E+05 5.63E+05 2.49E-03 2.49E-03 4.43 4.43

Figure 11 Figure

A 400- 400 ^ Blank Blank 400- B 400 OBlank A Blank I llsotype Isotype Isotype Isotype □ PF2566 PF2566 □MP4-1 MP4-1 300- 300 300- 300 □ 2D10 2D10 964 9B4 -M 10A3 10A3 =3

A O 200- 200 o 200- 200 u u

/ 100- 100 100- 100

i

f V 0o ..■I '"I '■I 0 0 "I T "I 10° 10° 101 10 ¹ 102 102 103 103 10° 10° 101 10 ¹ 102 102 103 103

Figure 22 Figure

1/3 1/3